The overall goal of Project 1 of the Research Program is to identify how airway epithelial cells initiate,[unreadable] intensify and modulate the inflammatory response through the generation of nitric oxide (NO) and reactive[unreadable] nitrogen species in the asthmatic airway. High output NO synthesis has been linked to airway inflammation[unreadable] in asthma. NO is produced by human airway epithelial cells (HAEC) that have increased NO synthase 2[unreadable] (NOS2) expression in asthma due to transcriptional activation of the gene. Our preliminary results show that[unreadable] interferon gamma (IFNgamma) induces, and interleukin-4 augments, NOS2 expression in HAEC in vitro through[unreadable] mechanisms that require physical interaction between activator prrotein (AP)-1 and IFNgamma-activated signal[unreadable] transducer and activator of transcription (STAT)-1 prior to DNA binding. In the context that asthma results[unreadable] from inflammatory processes that injure or modify airway function, excessive NO may participate in the[unreadable] pathogenesis of asthma through reactive nitrogen species formation and subsequent oxidation of proteins,[unreadable] modulating their biologic functions. In support of this, nitrated proteins are increased in the asthmatic airway.[unreadable] Using an innovative proteomic approach, we have identified tyrosine nitration of specific proteins in lung[unreadable] epithelial cells after NOS2 induction, and in the human airway, e.g. Mn superoxide dismutase (MnSOD) and[unreadable] catalase. Our preliminary data show that reactive nitrogen and oxygen species lead to loss of catalase and[unreadable] MnSOD activity, and consequent apoptosis. Taken together, we hypothesize that NO synthesis is increased[unreadable] in asthma due to NOS2 gene induction by IFNgamma utilizing AP-1 and STAT-1 signaling mechanisms in the[unreadable] airway epithelial cell, and that the generation of excess NO leads to protein oxidation modifying protein[unreadable] functions and contributing to airway injury/inflammation. To test this hypothesis, we will (1) define the[unreadable] regulation of NOS2 gene expression using HAEC in culture, and airway cells freshly obtained from asthmatic[unreadable] and healthy control lungs, and (2) determine the role of NO in asthma by a proteomic approach to identify[unreadable] nitrated proteins before and after NOS2 induction in HAEC in culture, in clinical samples from asthmatics in[unreadable] comparison to controls before and after an allergen challenge, and in an allergen-induced airway[unreadable] inflammation mouse model. Quantitation of biological tyrosine nitration/oxidation will be performed in[unreadable] collaboration with Project 3. Experiments to assess biologic effects of NO/reactive nitrogen species on[unreadable] extracellular matrix formation will be done in concert with Project 2. All Cores will be used extensively by[unreadable] Project 1. Together, these studies will provide a valuable comprehensive picture of the mechanisms that[unreadable] control NO synthesis in the human airway epithelium, alterations in those mechanisms that lead to asthma,[unreadable] and functional consequences of increased NO in the airway.